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Citrate Utilization Test- Principle, Procedure and Result Interpretation
Objectives of Citrate Utilization Test
The purpose of this test is to identify organisms capable of using sodium citrate as the sole carbon source and inorganic ammonium salts as the sole nitrogen source. The test is part of a series referred to as IMViC (indole, methyl red, Voges-Proskauer, and citrate), which is used to differentiate Enterobacteriaceae from other gram-negative rods.
Principle of Citrate Utilization Test
Bacteria that can grow on this medium produce an enzyme, citrate-permease, capable of converting citrate to pyruvate. Pyruvate can then enter the organism’s metabolic cycle for the production of energy. Bacteria capable of
growth in this medium use the citrate and convert ammonium phosphate to ammonia and ammonium hydroxide, creating an alkaline pH. The pH change turns the bromthymol blue indicator from green to blue.
NH4H2PO4 (1 g), K2HPO4 (1 g), NaCl (5 g), sodium citrate (2 g), MgSO4 (0.2 g), agar (15 g), bromthymol blue (0.08 g), per 1000 mL, pH 6.9.
Procedure of Citrate Utilization Test
- Inoculate Simmons citrate agar lightly on the slant by touching the tip of a needle to a colony that is 18 to 24 hours old. Do not inoculate from a broth culture, because the inoculum will be too heavy.
- Incubate at 35°-37°C for up to 7 days.
- Observe for growth and the development of blue color, denoting alkalinization.
Result Interpretation of Citrate Utilization Test
Positive: Growth on the medium, with or without a change in the color of the indicator. Growth typically results in the bromthymol blue indicator turning from green to blue.
Negative: Absence of growth.
Limitations of Citrate Utilization Test
Some organisms are capable of growth on citrate and do not produce a color change. Growth is considered a positive citrate utilization test, even in the absence of a color change.
Positive: Enterobacter aerogenes (ATCC13048)—growth, blue color
Negative: Escherichia coli (ATCC25922)—little to no growth, no color change